Activity-Based Photosensitizers with Optimized Triplet State Characteristics Toward Cancer Cell Selective and Image Guided Photodynamic Therapy

Activity-based theranostic photosensitizers are highly attractive in photodynamic therapy as they offer enhanced therapeutic outcome on cancer cells with an imaging opportunity at the same time. However, photosensitizers (PS) cores that can be easily converted to activity-based photosensitizers (aPSs) are still quite limited in the literature. In this study, we modified the dicyanomethylene-4H-chromene (DCM) core with a heavy iodine atom to get two different PSs (DCMO-I, I-DCMO-Cl) that can be further converted to aPS after simple modifications. The effect of iodine positioning on singlet oxygen generation capacity was also evaluated through computational studies. DCMO-I showed better performance in solution experiments and further proved to be a promising phototheranostic scaffold via cell culture studies. Later, a cysteine (Cys) activatable PS based on the DCMO-I core (DCMO-I-Cys) was developed, which induced selective photocytotoxicity along with a fluorescence turn-on response in Cys rich cancer cells.


Photophysical Characterization
Unless otherwise stated, all the absorbance and fluorescence measurements were made according to the following procedure. In a 3-mL quartz cell of 1-cm optical length, 10 μM of the stock solution of DCM, DCMO-I and I-DCMO-Cl was dissolved in DMSO-PBS buffer (10 mM, pH 7.4, 1:1, v/v) at 37 °C. In all fluorescence measurements, both excitation and emission slit widths were set to 10 nm.

Fluorescence Quantum Yield Calculation
The fluorescence quantum yield measurements of DCMO-I and I-DCMO-Cl were carried out using B-DCM-N (quantum yield was reported as 59.58% in DCM) 5 as the reference. For both measurements, the samples were dissolved in DMSO (10 mM, containing 1% PBS) and absorbance values of samples at their excitation wavelengths were kept less than 0.1.
The fluorescence quantum yield (ϕF) was calculated by using the following formula: where QYsample and QYstandard are the fluorescence quantum yields of the sample and the standard (B-DCM-N), respectively. Fsample and Fstandard are the integrated fluorescence emission of the sample, Asample and Astandard represent the absorbance of the sample and standard at their respective excitation wavelengths. Measurements were carried out in 1 cm quartz cuvettes with a total sample volume of 3 mL.

HPLC Analysis:
HPLC analysis was performed using RP-HPLC System with UV-Vis detection and a reversed-phase C18 column (4 μm, 4.6 × 150 mm). The data collect and analysis was carried out using the Chemstation software. The oven temperature was maintained at 25 ̊C, the injection volume was 20 µL and the flow rate was 1.0 mL/min with detection wavelength at 480 nm.The separation program of gradient elution was as follows ; where solvent A was water with 0.1% TFA and solvent B was acetonitrile.

Computational Studies:
TDDFT benchmark A benchmark of density functionals and basis sets was conducted to determine the method of choice for the present study. The data is available Table S4-S5 and a summary is presented in Table S6. In the latter, we report the error made by the respective methods for the energy difference between bright singlet states of DCMO-I in DMSO compared to the experimental spectrum. We notice that the topology of the excited states does not change from one method to another (data not shown), and that only the energies and ordering of the states vary. Only the rangeseparated double-hybrid functionals with spin-component or spin-opposite scaling SCS/SOC-wPBEPP86 reproduces accurately the energy difference between the ground state (S0) and the first excited singlet state (S1), with an error of 0.023 eV. The difference in energy between S1 and S2, as well as that between S2 and the third bright state (i.e., S3 or S4 depending on the method, further referred to as S3/4), are significantly over-estimated by the method with errors of 0.424 and 0.382 eV, respectively. The double-hybrid range separated functional with spin-component scaling B97X-2 significantly underestimates the S0-S1 energy difference and gives an unsatisfactory picture of the excited states. Other range-separated and double-hybrid range-separated functionals tested in this study were also found unsatisfactory. The hybrid functionals B3LYP, PBE0, and TPSSH, however, yield a fair prediction of the energy of S1 relative to the ground state, and offer the best energy difference between excited states over the methods tested in this benchmark. B3LYP slightly over-estimates the energy gap between S0 and S1 by 0.281 eV. The difference between S1-S2 and S2-S3/4 is, however, very well-reproduced by the method with errors compared to experiments of only 0.003 and -0.069 eV, respectively. Additionally, we find that B3LYP reproduces fairly well the solvent-induced red-shift on the absorption spectrum compared to other functional, while the choice of basis set only has a limited impact on the results (Table S4-S5).
Based on a Wigner distribution around the ground state minimum geometry of DCMo-I at 300 K, we calculated the absorption spectrum of the molecule at the B3LYP/aug-cc-pVDZ/cc-pVTZ-DK (iodine)/CPCM(DMSO) level and present it in Figure S14. The experimental and theoretical spectra are normalized to the intensity of the brightest peak, corresponding to the S0-S1 absorption. The sticks corresponding to the absorption in the minimum geometry are also shown in the figure, and a decomposition of the spectrum is given in terms of the different singlet states. Because S3 and S4 are very close in energy, their order is often inverted with distortion of the geometry through the Wigner distribution. One of them, nevertheless, is always dark while this other one is bright. The contribution of the third bright state is therefore referred to as S3/4. Considering vibrational distortion at 300 K broadens and red shifts all peaks, resulting in a predicted spectrum in fairly good agreement with the experimental one.

Table S4
Benchmark of the energy (in eV) of the first 6 singlet states of DCMO-I. The basis set is given for H, C, N, O, and that of iodine was (aug-)cc-pVTZ-DK in all cases, with diffuse functions added consistently with the main basis set. For aug-cc-pVDZ*, diffuse functions on iodine were omitted.The time is the user time on 4 cores on Intel(R) Xeon(R) Gold 6258R CPU@2.70GHz. The oscillator strength for each state is given in parenthesis. For comparison, the first three bright states of DCMO-I in DMSO are found experimentally at 1.9 eV, 2.9 eV, and 3.1 eV. "g.p." stands for gas phase.         Table S 11 Square modulus of spin-orbit coupling matrix elements (in cm -1 ; bold font) between singlet and triplet states of I-DCMO-Cl. The energy of each state is also given in eV and the oscillator strength of the singlet states in indicated as "f".